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Derivation of feeder-free human extended pluripotent stem cells.

ABSTRACT: Human extended pluripotent stem cells (EPSCs), with bidirectional chimeric ability to contribute to both embryonic and extraembryonic lineages, can be obtained and maintained by converting conventional pluripotent stem cells using chemicals. However, the transition system is based on inactivated mouse fibroblasts, and the underlying mechanism is not clear. Here we report a Matrigel-based feeder-free method to convert human embryonic stem cells and induced pluripotent stem cells into EPSCs and demonstrate the extended pluripotency in terms of molecular features, chimeric ability, and transcriptome. We further identify chemicals targeting glycolysis and histone methyltransferase to facilitate the conversion to and maintenance of feeder-free EPSCs. Altogether, our data not only establish a feeder-free system to generate human EPSCs, which should facilitate the mechanistic studies of extended pluripotency and further applications, but also provide additional insights into the transitions among different pluripotent states.

SUBMITTER: Zheng R

PROVIDER: S-EPMC8282469 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Derivation of feeder-free human extended pluripotent stem cells.

Zheng Ran R Geng Ting T Wu Dan-Ya DY Zhang Tianzhe T He Hai-Nan HN Du Hai-Ning HN Zhang Donghui D Miao Yi-Liang YL Jiang Wei W

Stem cell reports 20210701 7

Human extended pluripotent stem cells (EPSCs), with bidirectional chimeric ability to contribute to both embryonic and extraembryonic lineages, can be obtained and maintained by converting conventional pluripotent stem cells using chemicals. However, the transition system is based on inactivated mouse fibroblasts, and the underlying mechanism is not clear. Here we report a Matrigel-based feeder-free method to convert human embryonic stem cells and induced pluripotent stem cells into EPSCs and de ...[more]

PMID: 34214484

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Project description:: This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000-200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%-95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs.SignificanceThis differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.

Dataset Information

Derivation of feeder-free human extended pluripotent stem cells.

Publications

Derivation of feeder-free human extended pluripotent stem cells.

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